Obesity and diabetes are globally increasing health problems and are associated with various diseases, particularly cardiovascular disease (CVD), obstructive sleep apnea, stroke, peripheral artery disease, microvascular complications and osteoarthritis.
About 250 million people worldwide suffer from diabetes, and by 2025 it is estimated that 380 million will have diabetes. Many have additional cardiovascular risk factors, including high/aberrant LDL and triglycerides and low HDL.
Cardiovascular disease accounts for about 50% of mortality in people with diabetes, and the morbidity and mortality rates relating to obesity and diabetes underscore the medical need for efficacious treatment options.
Preproglucagon is a 158 amino acid precursor polypeptide that is differentially processed in the tissues to form a number of structurally related proglucagon-derived peptides, including glucagon (Glu), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and oxyntomodulin (OXM). These molecules are involved in a wide variety of physiological functions, including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, as well as regulation of food intake.
Glucagon is a 29-amino acid peptide that corresponds to amino acids 53 to 81 of pre-proglucagon, and has the following amino acid sequence (written using conventional three-letter amino acid abbreviations): His Ser-Gln-Gly-Thr-Phe-Thr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Lue-Met-Asn-Thr (SEQ ID NO:86). The amino acid sequence of glucagon written using conventional one-letter amino acid abbreviations is: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO:86).
Oxyntomodulin (OXM) is a 37-amino acid peptide which includes the complete 29-amino acid sequence of glucagon with an octapeptide carboxy-terminal extension. The latter extension consists of amino acids 82 to 89 of pre-proglucagon, having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala and termed “intervening peptide 1” or IP-1; the full sequence of human oxyntomodulin is thus His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg- Asn-Asn-Ile-Ala (SEQ ID NO:87).
The major biologically active fragment of GLP-1 is produced as a 30-amino acid, C-terminally amidated peptide that corresponds to amino acids 98 to 127 of pre-proglucagon.
Glucagon helps maintain the level of glucose in the blood by binding to glucagon receptors on hepatocytes, causing the liver to release glucose—stored in the form of glycogen—through glycogenolysis. As these stores become depleted, glucagon stimulates the liver to synthesize additional glucose by gluconeogenesis. This glucose is released into the bloodstream, preventing the development of hypoglycemia. Additionally, glucagon has been demonstrated to increase lipolysis and decrease body weight.
GLP-1 decreases elevated blood glucose levels by improving glucose-stimulated insulin secretion. It also promotes weight loss, primarily through decreasing food intake.
Oxyntomodulin is released into the blood in response to food ingestion and in proportion to meal calorie content. The mechanism of action of oxyntomodulin is not well understood. In particular, it is not known whether the effects of the hormone are mediated exclusively through the glucagon receptor and the GLP-1 receptor, or through one or more as-yet unidentified receptors.
Other peptides have been shown to bind and activate both the glucagon and the GLP-1 receptor (see, e.g., Hjort et al, Journal of Biological Chemistry, 269, 30121-30124, 1994) and to suppress body weight gain and reduce food intake (WO 2006/134340; WO 2007/100535; WO 2008/101017).
Among the amino acid residues in the sequence of native glucagon, the residues and positions 3 and 4 (Gln and Gly, respectively, in native human glucagon), respectively, appear to be rather generally regarded as not being susceptible to substitution without loss of physiological activity. However, the present inventors believe that the observed, relatively facile deamidation of the Gln residue at position 3 of native glucagon is to a large extent associated with the presence of the sterically small Gly residue in the neighbouring 4 position. It would thus be highly desirable to be able to substitute, inter alia, one or both of the residues at positions 3 and 4 and still be able to achieve peptides possessing useful physiological activity.